1. Field of the Invention (Technical Field)
Embodiments of the present invention relates to the use of membrane substrates having nano-sized pores within which capturing elements of a biological or chemical nature are immobilized for the detection of target biological objects, or analytes, using changes in ionic conductance through the nanopores as a result of hybridization between the capturing elements and the target biological objects. The present invention also relates to the use of membranes having nano-sized pores in which complimentary ligands are immobilized for the detection of complimentary target analytes through hybridization wherein detection utilizing changes in ionic conductance through the nanopores as a result of surface charge change from hybridization.
2. Background Art
DNA microarray chips for the detection of oligonucleotides, as known in the art, rely on such detection means as the fluorescence detection of modified DNA oligonucleotides. Controlling the mass transport of specific species through nanopores by means of UV light, pH of solution, charge, and size of an ion has been explored. Biosensors and separation membranes that are based on selective nanopores have been studied, and technological advances have made it possible to manufacture nanopores with dimensions comparable to the sizes of biological polymers such as short DNA and peptides. Advantage has been made of such an approach and single nanopores have been used to resolve the sequences of individual DNA molecules linked to a degree of partial pore blockage by the DNA.
Detection and separation of DNA using nanoporous alumina filters has also been accomplished. U.S. patent application Ser. No. 11/235,824 filed Sep. 26, 2005 disclosed detection and separation apparatuses and methods. U.S. patent application Ser. No. 11/235,824 is incorporated herein by reference.
Alternative electrical detection schemes usually involve electrochemical methods that also require complicated detection means and methods. Measuring changes in ionic conductance through a nanopore or a nanoporous membrane presents an opportunity to more easily detect target biological objects.
Applications of nanotechnology are particularly versatile when it comes to chemical and biochemical sensors. Bioaffinity interactions such as DNA-DNA and antigen-antibody are typically employed for identification of the presence of a particular DNA sequence in a sample, for detection of microbial and viral species. The mechanism of detecting such an interaction with untagged biochemical analytes can vary broadly and utilize the change of mass, volume, charge, optical, or other properties of the analytes.
Because of a large surface/volume ratio in nanoporous materials, the ionic conductance through the pores is greatly affected by the ions' interaction with the walls. This interaction can manifest itself through different mechanisms, most commonly via the “volume exclusion” and the “surface charge” mechanisms. Previously, we have demonstrated the utilization of the volume exclusion mechanism for DNA detecting. In this mechanism, small diameter pores get “clogged” as a result of binding targeted DNA onto single-stranded DNA (ss-DNA) covalently attached to the walls. The result is detected by a drop in ionic flux through the pores. The observed effect was strongly dependent on the pore diameter: almost nonexistent with 200 nm pores and reaching in excess of 50% in 20 nm or smaller pores.
Note that where the discussion herein refers to a number of publications by author(s) and year of publication, that, due to recent publication dates, certain publications are not to be considered as prior art vis-à-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.